BLEEDING EPISODES

Field of the Invention

The present invention provides a method and compounds for treatment and prophylaxis of bleeding episodes, for example bleeding episodes in subjects suffering from bleeding disorders such as haemophilia.

Background to the Invention

Bleeding episodes may be either spontaneous or triggered by an event, for example, surgery, trauma, pregnancy or childbirth. Bleeding episodes occur frequently in patients suffering from a bleeding disorder or liver disease.

Haemophilia is an example of an X linked bleeding disorder which is caused by mutations in the genes encoding for clotting factor VIII (Haemophilia A) or clotting factor IX (Haemophilia B). The prevalence of both disorders is 1 in 5000 male births for Haemophilia A and 1 in 30000 male births for Haemophilia B. Both factor VIII and factor IX are integral components of the intrinsic contact clotting pathway and are required for normal haemostasis. Individuals may have mild, moderate or severe disease, defined by factor levels of 6-30%, 2-5%, and less than 1 %, respectively. Excessive bleeding episodes are seen in mild haemophilia patients following surgery or major trauma, while frequent episodes of spontaneous bleeding or excessive bleeding post minor trauma are seen in severe haemophilia (Franchini M. Blood Transfus. 2013;11 (2):178-82).

The replacement of the deficient factor VIII or IX through plasma-derived or recombinant concentrates is the main treatment strategy for haemophilia (Franchini M., 2013; Coppola A. Journal of Blood Medicine. 2010. p. 183). However, inhibitors or alloantibodies against plasma concentrate factor VIII and IX develop in 20-30% of patients with severe Haemophilia A and to a lesser extent in Haemophilia B (Farrugia A, Hermans C, Franchini M. Haemophilia. 2015 May;21 (3):307-9; Osooli M, Berntorp E. J Intern Med [Internet]. 2015 Jan;277(1 ):1-15). Recombinant and plasma-derived activated factor VII (FVIIa) proteins are currently used to treat bleeding episodes in patients with haemophilia who develop inhibitors or alloantibodies following factor VIII or factor IX replacement therapy. FVIIa activates clotting factor X and can induce thrombin generation in the absence of factor VIII and factor IX. However, FVIIa is an extremely potent activator of factor X and therefore an extremely potent activator of clotting. As such, there is a risk of thrombotic complications, including disseminated intravascular coagulation following treatment with FVIIa. Alternative strategies to resolve or prevent bleeding episodes in haemophilia are therefore required, particularly in haemophilia patients who develop inhibitors or alloantibodies following factor VIII or factor IX replacement therapy.

During platelet activation, platelet basic protein is cleaved at the N-terminus to form connective tissue activating peptide III (CTAPIII), beta-thromboglobulin and neutrophil-activating peptide (NAP-2) (Brandt, E., Petersen, F., Ludwig, A., Ehlert, J.E., Bock, L. and Flad, Hans-D. April 2000. Journal of Leukocyte Biology. Vol. 67). These derivatives of platelet basic protein are referred to collectively herein as "platelet-derived beta-thromboglobulin". CTAPIII is the main isoform in platelet-derived beta-thromboglobulin. It is stored in high concentrations by platelets in their alpha granules (Blair P, Flaumenhaft R. Blood Rev. 2009 Jul;23(4):177-89). During platelet activation at sites of vascular injury, CTAPIII is released into the extracellular environment (Kaplan KL, Owen J. Blood. 1981 Feb;57(2):199-202)). CTAPIII may be cleaved to form NAP-2 (Car, B.D., Baggiolini M. and Walz, A. Biochem. J. 1991 ; 275, 581 -584). The physiological function of CTAPIII and the other derivatives of platelet basic protein has been poorly characterised to date.

Summary of the Invention

According to a first aspect of the present invention there is provided a derivative of platelet basic protein selected from one or more of connective tissue activating peptide III (CTAPIII), beta-thromboglobulin and neutrophil-activating peptide (NAP-2) for use as a medicament Also provided is a derivative of platelet basic protein selected from one or more of connective tissue activating peptide III (CTAPIII), beta-thromboglobulin and neutrophil-activating peptide (NAP-2) for use in treatment or prophylaxis of a bleeding episode in a subject in need thereof.

Further provided is a method for treating or preventing a bleeding episode in a subject in need thereof comprising administering a derivative of platelet basic protein selected from one or more of connective tissue activating peptide III (CTAPIII), beta-thromboglobulin and neutrophil-activating peptide (NAP-2) to a subject in need thereof.

Also provided is use of a derivative of platelet basic protein selected from one or more of connective tissue activating peptide III (CTAPIII), beta-thromboglobulin and neutrophil-activating peptide (NAP-2) in preparation of a medicament for treatment or prevention of a bleeding disorder in a subject in need thereof.

Also provided is a pharmaceutical composition comprising a derivative of platelet basic protein selected from one or more of connective tissue activating peptide III (CTAPIII), beta-thromboglobulin and neutrophil-activating peptide (NAP-2) and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition may also comprise a secondary therapeutic compound used in the treatment or prevention of a bleeding episode.

The present inventors have demonstrated that N-terminal derivatives of platelet basic protein such as connective tissue activating peptide III (CTAPIII), possess procoagulant activity and may therefore be used to promote clotting in subjects.

The derivative of platelet basic protein may be administered to the subject prophylactically to prevent a bleeding episode in a subject at risk of a bleeding episode, for example, a subject who is undergoing surgery or who suffers from a bleeding disorder or liver disease. Alternatively the derivative of platelet basic protein may be administered on demand to resolve or treat a bleeding episode in the subject. In certain embodiments the bleeding episode is selected from one or more of the group consisting of a bleeding episode associated with a bleeding disorder (such as Haemophilia A (International Classification of Diseases (ICD) 286.0), Haemophilia B (ICD 286.1 ), Von Willebrand Disease (ICD 286.4), Haemorrhagic disorder due to intrinsic anticoagulants (ICD 286.5), Disseminated intravascular coagulation (ICD 286.6), Acquired coagulation factor deficiency (ICD 286.7), FXIII deficiency (ICD 286.3), Factor V deficiency, Factor X deficiency (mild/moderate), Factor VII deficiency, Hypofibrinogenemia, Factor II deficiency (ICD 286.9) (coagulation defects, other), Glanzmann thrombasthenia, Thrombocytopenia (low platelets) and purpura (ICD 287.3, ICD 287.4)), bleeding due to trauma, surgery, pregnancy or childbirth, bleeding due to liver disease or cirrhosis of the liver, bleeding due to HIV, coagulopathy due to leukemic variant M3, bleeding due to vitamin K deficiency, bleeding resulting from medications that thin the blood, such as aspirin, heparin and warfarin, and unspecified bleeding (ICD 287.9).

Where the subject suffers from a bleeding disorder, the bleeding disorder may therefore be caused by mutations in genes encoding for a component of the blood clotting process, such as a clotting factor or platelets. In particular, the bleeding disorder may be caused by mutations in genes encoding one or more factors selected from the group consisting of factor VIII, factor IX, von Willebrand factor (VWF), factor II, factor V, factor VII, factor X and factor XIII. The subject may be missing the factor in question, have reduced levels of the factor or have a defective factor. Typically, the subject will have some functioning factor X, i.e. factor X will not be completely absent in the subject.

Typically, the bleeding disorder may be caused by mutations in genes encoding for factor VIII (Haemophilia A) or factor IX (Haemophilia B). Accordingly, in certain embodiments the bleeding disorder is haemophilia, more specifically Haemophilia A or Haemophilia B. In particular, in certain embodiments the bleeding disorder is haemophilia (Haemophilia A or Haemophilia B) where the subject develops inhibitors or alloantibodies following factor VIII or factor IX replacement therapy.

The present inventors have demonstrated that the derivative of platelet basic protein, such as CTAPIII, can advantageously induce clotting in the absence of factor VIII and factor IX. Specifically, a derivative of platelet basic protein, such as CTAPIII, can induce clotting through activation of factor X and this pathway is independent of factor VIII and factor IX. The inventors have shown that a derivative of platelet basic protein, such as CTAPIII, can recover thrombin generation in factor VIII and factor IX immunodepleted plasmas that are similar to plasma derived from patients with haemophilia A and B, respectively. The use of a derivative of platelet basic protein, such as CTAPIII, therefore provides a novel treatment for subjects with haemophilia, particularly Haemophilia A or Haemophilia B. It is a less potent activator of factor X than activated factor VII, thus reducing the risk of thrombotic complications associated with activated factor VII, such as disseminated intravascular coagulation. The use of a derivative of platelet basic protein, such as CTAPIII, therefore provides an alternative treatment strategy for subjects with haemophilia (Haemophilia A or Haemophilia B) who develop inhibitors or alloantibodies following factor VIII or factor IX replacement therapy.

The derivative of platelet basic protein is selected from one or more of the group consisting of connective tissue activating peptide III (CTAPIII), beta- thromboglobulin and neutrophil-activating peptide (NAP-2). In certain embodiments the derivative of platelet basic protein comprises a combination of connective tissue activating peptide III (CTAPIII), beta-thromboglobulin and neutrophil-activating peptide (NAP-2). Typically the derivative of platelet basic protein comprises, consists essentially of or consists of CTAPIII. The derivative of platelet basic protein, such as CTAPIII, may be platelet-derived (i.e. obtained from platelets) or a recombinant form thereof, or a combination thereof. Where the derivative of platelet basic protein is recombinant, the recombinant derivative of platelet basic protein may comprise, include or consist of an amino acid sequence which has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence of the wild type derivative of platelet basic protein. Typically the recombinant derivative of platelet basic protein activates factor X, preferably either at the same level or at an increased level when compared to the wild type derivative of platelet basic protein. The amino acid sequence of wild type CTAPIII is provided below as SEQ ID NO:1 :

NLAKGKEESLDSDLYAELRCMCIKTTSGIHPKNIQSLEVIGKGTHCNQVEVIATLK DGRKICLDPDAPRIKKIVQKKLAGDESAD

Accordingly, typically recombinant CTAPIII will have an amino acid sequence which has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with SEQ ID NO:1.

In certain embodiments, the recombinant derivative of platelet basic protein comprises one or more mutations or amino acid substitutions. The mutations or substitutions may prevent or inhibit cleavage of the derivative of platelet basic protein. Advantageously this may extend the half life of the derivative of platelet basic protein. Typically, the recombinant derivative of platelet basic protein comprises one, two, three, four, five, six, seven, eight, nine or ten mutations or amino acid substitutions.

Where the derivative of platelet basic protein is CTAPIII, typically the one or more mutations or amino acid substitutions prevent or inhibit cleavage of CTAPIII to form NAP-2, i.e. the cleavage site where CTAPIII is cleaved to form NAP-2 is removed. In that case, the recombinant CTAPIII may include one or more mutations or amino acid substitutions at, or close to, the region of SEQ ID NO:1 where CTAPIII is cleaved to form NAP-2. CTAPIII is cleaved by chymotrypsin and cathepsin G. Both proteinases cleave preferentially the bond between amino acids 15 (Tyr) and 16 (Ala) of CTAPIII (Car et al.). Accordingly, in certain embodiments recombinant CTAPIII comprises one or more mutations in the region of amino acid residues 10-21 , 1 1 -20, 12-19, 13-18, 14-17 or 15-16 of SEQ ID NO:1. In certain embodiments recombinant CTAPIII comprises one, two or three amino acid substitutions selected from the group consisting of D13A (i.e. amino acid D at position 13 is replaced with A), L14A and Y15A. Typically the recombinant CTAPIII comprises the amino acid substitution Y15A. Additionally the recombinant CTAPIII may comprise the amino acid substitution D13A and/or the amino acid substitution L14A. Preferably when the amino acid sequence of the derivative of platelet basic protein is modified by way of deletion or substitution of any of the amino acid residues contained therein, these changes have no negative effect on the functional activity of the modified derivative of platelet basic protein when compared to the wild type derivative of platelet basic protein. Typically the one or more mutations do not change the overall 3D structure of the derivative of platelet basic protein. This may be assessed using SWISS-PROT.

The derivative of platelet basic protein, such as CTAPIII, may be administered in combination with one or more additional treatments for a bleeding episode.

The bleeding episode may be mild, moderate or severe. Brief Description of the Figures

The present invention will now be described with reference to the following examples which are provided for the purpose of illustration and are not intended to be construed as being limiting on the present invention wherein:

Figure 1 : Effect of platelet derived β-thromboglobulin on Tissue Factor (TF) dependent thrombin generation and the prothrombin time (mean+SEM). Thrombin generation (1 pM Tissue Factor, 4μΜ phospholipids) and the PT were measured in platelet poor plasma incubated with increasing concentrations of β-thromboglobulin. (A) Mean thrombin generation curves in normal pooled plasma incubated with 0 μ9/ιηΙ (open circles), 5μ9/ιηΙ (open square), 5C^g/ml (closed circle), and 10C^g/ml β-thromboglobulin (closed square). (B) Lagtime, (C) time to peak, (D) peak thrombin generation, and (E) endogenous thrombin potential of thrombin generation, (f) Prothrombin time in the presence and absence of β-thromboglobulin. Data was analysed by paired t test relative to 0 μg/ml β-thromboglobulin. ^* P-values less than 0.05 were considered statistically significant.

Figure 2: Effect of platelet derived β-thromboglobulin on TF independent thrombin generation and the activated partial thromboplastin time (mean+SEM). Thrombin generation (4μΜ phospholipids) and the APTT were measured in normal pooled plasma incubated with β- thromboglobulin. (A) Mean thrombin generation curves in normal pooled plasma incubated with 0μg/ml (open circles) and 50μg/ml β- thromboglobulin (closed circle). (B) Lagtime, (C) time to peak, (D) peak thrombin generation, and (E) endogenous thrombin potential of thrombin generation. (F) Activated partial thromboplastin time in the presence and absence of β-thromboglobulin. Data was analysed by paired t test relative to 0 μg/ml β-thromboglobulin. ^* P-values less than 0.05 were considered statistically significant.

Figure 3: Platelet derived β-thromboglobulin induces TF independent thrombin generation in Factor VIII and Factor IX deficient plasma (mean + SEM). Thrombin generation (4μΜ phospholipids) and the APTT were measured in Factor IX and Factor VIII deficient plasma incubated with β- thromboglobulin. (A) Mean thrombin generation curves in Factor IX deficient plasma in the presence of 0μg/ml and 50μg/ml beta- thromboglobulin. (B) Peak thrombin generation in Factor IX deficient plasma incubated with 0μg/ml and 50 μg/ml β-thromboglobulin. (C) Mean thrombin generation curves in Factor VIII deficient plasma in the presence of 0 μg/ml and 50 μg/ml β-thromboglobulin. (D) Peak thrombin generation in Factor VIII deficient plasma incubated with 0μg/ml and 50μg/ml β- thromboglobulin. (E) APTT in factor VIII deficient plasma in the absence and presence of C^g/ml and 50 μ9/ιηΙ β-thromboglobulin. (F) Comparison of the effects of β-thromboglobulin (50 μ9/ηηΙ ± TF and FVIIa inhibitory antibodies), Platelet Factor 4 (50 μg/ml), and Factor IXa (50 μg/ml) on thrombin generation in Factor VIII deficient plasma. Data was analysed by paired t test or repeated measure ANOVA with Tukey's multiple comparison post test. ^* P-values less than 0.05 were considered statistically significant.

Figure 4: Effect of platelet derived β-thromboglobulin on thrombin generation in Factor V and Factor X deficient plasma (mean + SEM). Thrombin generation (4μΜ phospholipids) was measured in Factor V deficient plasma (± 0, 2, 5 nM FVa) and Factor X deficient plasma (± 0, 0.5, 5 nM FXa) in the presence or absence of β-thromboglobulin (50 μg/ml). (A) Mean thrombin generation curves in Factor V deficient plasma. (B) Lagtime to thrombin generation in Factor V deficient plasma supplemented with 2 or 5 nM FVa (± 50μg/ml β-thromboglobulin). (C) Mean thrombin generation curves in Factor X deficient plasma. (D) Lagtime to thrombin generation in Factor X deficient plasma supplemented with 0.5 or 5 nM FXa (± 50μg/ml β-thromboglobulin). Data was analysed by paired t test relative to 0 μg/ml β-thromboglobulin. *P- values less than 0.05 were considered statistically significant.

Figure 5: Platelet derived β-thromboglobulin interacts with Factor X (mean + SEM). The interaction between β-thromboglobulin and Factor X was studied using a chromogenic assay of Factor X activation and surface plasmon resonance. (A) Chromogenic assay of Factor X activation. Factor Xa (100nM), Factor X (2μΜ), β-thromboglobulin (200ug/ml), Factor X plus β-thromboglobulin (2μΜ + 200μg/ml), and Factor X plus Factor IXa (2μΜ + 200μg/ml) were incubated for 1 hour followed by the addition of a Factor Xa specific chromogenic substrate (1 mg/ml). Substrate cleavage was measured by absorbance at 405nm after 20 minutes. (B) Chromogenic assay of Factor II activation. Factor I la (100nM), Factor II (2μΜ), β- thromboglobulin (200μg/ml), and Factor II plus β-thromboglobulin (2μΜ + 20C^g/ml) were incubated for 1 hour followed by the addition of a Factor I la specific chromogenic substrate (1 mg/ml). Substrate cleavage was measured by absorbance at 405nm after 20 minutes. (C) Surface plasmon resonance sensogram plot showing the binding interactions of β- thromboglobulin (closed circle:, 20μg/ml, open circle: l Opg/ml, closed square; 5μg/ml) with immobilised factor X. (D) Surface plasmon resonance sensogram plot showing the binding interactions of Factor IXa (closed circle: 4μg/ml, open circle: 2μg/ml, closed square; ^g/ml) with immobilised factor X. (E) Association and dissociation kinetics for β- thromboglobulin and Factor IXa interactions with immobilised Factor X, measured by surface plasmon resonance. Data was analysed by repeated measures ANOVA with Tukey's multiple comparison post test. *P-values less than 0.05 were considered statistically significant. Detailed Description of the Invention

The inventors have shown that platelet derived β-thromboglobulin (approximately 95% of which is connective tissue activating peptide III (CTAPIII)) possesses procoagulant activity and that platelet derived β-thromboglobulin can induce clotting through activation of factor X independently of factor VIII and factor IX. The inventors have shown that platelet derived β-thromboglobulin can recover thrombin generation in factor VIII and factor IX immunodepleted plasmas that are similar to plasma derived from patients with haemophilia A and B, respectively. Platelet derived β-thromboglobulin is a less potent activator of factor X than activated factor VII, thus reducing the risk of thrombotic complications associated with activated factor VII, such as disseminated intravascular coagulation. Platelet derived β-thromboglobulin therefore provides an alternative treatment strategy for subjects with haemophilia (Haemophilia A or Haemophilia B) who develop inhibitors or alloantibodies following factor VIII or factor IX replacement therapy.

The derivative of platelet basic protein, such as CTAPIII, may be administered alone or as a pharmaceutical composition, which will generally comprise a suitable pharmaceutically acceptable excipient, diluent or carrier selected depending on the intended route of administration. Examples of suitable pharmaceutical carriers include water, glycerol, ethanol and other GRAS reagents. The derivative of platelet basic protein, such as CTAPIII, may be administered to a patient in need of treatment, typically a patient suffering from a bleeding disorder or liver disease and/or at risk of a bleeding episode. Administration may be via any suitable route, for example parenterally by injection or infusion. Examples of routes for parenteral administration include, but are not limited to, intravenous, intracardial, intraarterial, intraperitoneal, intramuscular, intracavity, subcutaneous, transmucosal, inhalation and transdermal. Routes of administration may further include topical and enteral, for example, mucosal (including pulmonary), oral, nasal and rectal.

The derivative of platelet basic protein, such as CTAPIII, is preferably administered to the subject in a therapeutically or prophylactically effective amount, this being sufficient to show benefit to the subject to whom the derivative of platelet basic protein, such as CTAPIII, is administered, for example, reduced bleeding, cessation of bleeding or a delay in or prevention of a bleeding episode. The actual dose administered, and rate and time-course of administration, will depend on, and can be determined with due reference to, the nature and severity of the bleeding episode or bleeding disorder which is being treated, as well as factors such as the age, sex and weight of the subject to be treated and the route of administration. Further due consideration should be given to the properties of the administered composition, as well as the site and rate of delivery. The derivative of platelet basic protein, such as CTAPIII, is administered to the subject for a time period and under conditions sufficient to treat or prevent a bleeding episode. Dosage regimens can include a single administration of the derivative of platelet basic protein, such as CTAPIII, or multiple administrative doses. The derivative of platelet basic protein, such as CTAPIII, can be administered simultaneously, sequentially or separately with other therapeutics and medicaments which are used for the treatment or prevention of bleeding episodes or bleeding disorders. The derivative of platelet basic protein, such as CTAPIII, and a second active compound may be administered together in a single composition. Alternatively, the derivative of platelet basic protein, such as CTAPIII, and a second active compound may be administered in separate compositions as part of a combined therapy. For example, the first compound may be administered before, after, or concurrently with the second compound.

Depending on the bleeding episode/disorder and its severity, the derivative of platelet basic protein, such as CTAPIII, may be administered (infused) prophylactically to a subject at risk of a bleeding episode to prevent a bleeding episode or the derivative of platelet basic protein, such as CTAPIII, may be administered on-demand to a subject undergoing a bleeding episode to treat or resolve the bleeding episode. For example, the derivative of platelet basic protein, such as CTAPIII, may be administered prophylactically to a subject prior to or whilst the subject is undergoing surgery where the subject is at risk of a bleeding episode, for example, where the subject is suffering from a bleeding disorder such as haemophilia. Alternatively, prophylaxis may involve infusion of the derivative of platelet basic protein, such as CTAPIII, on a regular schedule in order to keep clotting levels sufficiently high to prevent spontaneous bleeding episodes. On-demand treatment involves treating bleeding episodes once they arise, for example, following trauma or surgery or during pregnancy or childbirth.

The derivative of platelet basic protein, such as CTAPIII, may also be provided in a form selected from the group consisting of an oral product, topical products, nasal sprays and fresh frozen plasma. The derivative of platelet basic protein, such as CTAPIII, may be administered at home or in a clinical setting.

The derivative of platelet basic protein, such as CTAPIII, may be platelet-derived. For example, washed platelet suspensions may be prepared from human donor blood and stimulated with a platelet agonist under stirring conditions to induce platelet activation and aggregation. The derivative of platelet basic protein may be prepared from the supernatant of the activated platelets by heparin-agarose affinity chromatography and high performance liquid chromatography. Examples of methods for obtaining derivatives of platelet basic protein, such as CTAPIII, from platelets are described in Castor C.W., Miller, J.W. and Walz, D.A. Proc. Natl. Acad. Sci. USA. 1983:Vol. 80, pp 765-769. Cell Biology.

The derivative of platelet basic protein, such as CTAPIII, may be a recombinant form, for example, a nucleic acid sequence for the derivative of platelet basic protein, such as CTAPIII, may be expressed in a suitable cell line, such as Chinese hamster ovary (CHO) tissue culture cells and isolated therefrom to provide recombinant CTAPIII. Recombinant proteins may offer higher purity and safety than plasma-derived or platelet-derived proteins.

Alternatively, treatment may be provided using gene therapy. In particular, the gene for the derivative of platelet basic protein, such as CTAPIII, may be inserted into a subject in need thereof using a suitable vector, such as an adeno- associated virus-8 vector. The transduced virus may be infused intravenously in a subject in need thereof, for example, a subject suffering from haemophilia.

Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by a person who is skilled in the art in the field of the present invention.

The term "bleeding episode" or "bleeding event" as used herein refers to extended bleeding in a subject. Bleeding episodes or events are characterised according to International Society on Thrombosis and Haemostasis (ISTH) criteria (Schulman, S. & Kearon, C. Journal of Thrombosis and Haemostasis. 2004.3:692-694). The bleeding episode may be triggered by an event, for example, it may occur after surgery, trauma, pregnancy or childbirth. Alternatively, it may be unspecified or spontaneous, i.e. without a known or identifiable cause. A bleeding episode may also be associated with a bleeding disorder, liver disease, cirrhosis of the liver, bleeding due to HIV, coagulopathy due to leukemic variant M3, bleeding due to vitamin K deficiency or bleeding resulting from medications that thin the blood, such as aspirin, heparin and warfarin. As used herein, a bleeding episode is considered to be associated with a bleeding disorder or other disease or condition where the subject is suffering from a bleeding disorder or other disease or condition. In particular, the bleeding episode may be caused by, or as a result of, the bleeding disorder or other disease or condition or the subject may be at an increased risk of a bleeding episode due to the bleeding disorder or other disease or condition.

The term "bleeding disorder" as used herein refers to a condition or disorder where there is an inability to form a proper blood clot. Improper clotting can be caused by defects in blood components such as platelets and/or clotting factors, such as factor VIII or factor IX. A subject suffering from a bleeding disorder is therefore more at risk of suffering from a bleeding episode. Symptoms of the bleeding disorder may be one or more of bleeding into joints, muscles and soft tissues, excessive bruising, prolonged, heavy menstrual periods (menorrhagia), unexplained nosebleeds and extended bleeding after minor cuts, blood draws or vaccinations, minor surgery or dental procedures. Bleeding disorders may include conditions as Haemophilia A (International Classification of Diseases (ICD) 286.0), Haemophilia B (ICD 286.1 ), Von Willebrand Disease (ICD 286.4), Haemorrhagic disorder due to intrinsic anticoagulants (ICD 286.5), Disseminated intravascular coagulation (ICD 286.6), Acquired coagulation factor deficiency (ICD 286.7), FXIII deficiency (ICD 286.3), Factor V deficiency, Factor X deficiency (mild/moderate), Factor VII deficiency, Hypofibrinogenemia, Factor II deficiency (ICD 286.9) (coagulation defects, other), Glanzmann thrombasthenia, Thrombocytopenia (low platelets) and purpura (ICD 287.3, ICD 287.4). The bleeding disorder, such as haemophilia, may be mild, moderate or severe.

The term "derivatives of platelet basic protein" as used herein is intended to refer to proteins which are derived or obtained by cleavage of platelet basic protein at the N-terminus particularly in humans, i.e. connective tissue activating peptide III (CTAPIII), beta-thromboglobulin and neutrophil-activating peptide (NAP-2). These may also be referred to herein as N-terminal deletion products or N- terminus truncated variants of platelet basic protein. The plasma levels of these proteins may be used as a marker of platelet activation and are measured by ELISA. The ELISAs are sold as platelet basic protein ELISA, NAP-2 ELISA, or beta-thromboglobulin ELISA.

The terms "platelet-derived beta-thromboglobulin", "platelet-derived 3TG" and "3TG" as used herein are intended to refer collectively to the group of proteins derived or obtained from cleavage of platelet basic protein at the N-terminus, i.e. the N-terminus derivatives of platelet basic protein CTAPIII, beta-thromboglobulin and NAP-2. Commercial preparations of platelet derived beta-thromboglobulin comprise 95-100% CTAPIII (Hoogewerf, A.J., Leone, J.W., Reardon, I.M., Howe, W.J., Asa, D., Heinrikson, R.L. and Ledbetter, S.R. The Journal of Biological Chemistry. Vol. 270, No. 7. Feb. 17 1995, 3268-3277).

The term "identity" or "sequence identity" as used herein means that at any particular position in two aligned sequences the amino acid residues at that position are identical in the aligned sequences. Sequence identity with respect to a wild type derivative of platelet basic protein and a recombinant form relates to the percentage of amino acid residues in the recombinant form which are identical with the residues of the corresponding wild type derivative of platelet basic protein after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage identity. Methods and computer programs for performing an alignment of two or more amino acid sequences and determining sequence identity are well known to the person skilled in the art. For example, the percentage of identity of two amino acid sequences can be readily calculated using algorithms e.g. BLAST.

The phrase "consists essentially of" as used herein may be understood to refer to the main component present, i.e. the component which is present in the greatest amount. Typically the presence or absence of other components will not affect the function or activity of the main component.

As used herein, the term "subject" refers to an animal, preferably a mammal and in particular a human. In a particular embodiment, the subject is a mammal, in particular a human. The term "subject" is interchangeable with the term "patient" as used herein.

Throughout the specification, unless the context demands otherwise, the terms "comprise" or "include", or variations such as "comprises" or "comprising", "includes" or "including" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. As used herein, terms such as "a", "an" and "the" include singular and plural referents unless the context clearly demands otherwise. Thus, for example, reference to "an active agent" or "a pharmacologically active agent" includes a single active agent as well as two or more different active agents in combination, while references to "a carrier" includes mixtures of two or more carriers as well as a single carrier, and the like.

EXAMPLES Methods

The effect of purified human 3TG on coagulation was determined using the prothrombin time (PT), activated partial thromboplastin time (APTT), calibrated automated thrombography (4μΜ phospholipids ± 1 pM tissue factor) and chromogenic FX and prothrombin activation assays. Mass spectrometry and surface plasmon resonance (SPR) were used to assess the composition of the purified 3TG preparation and to measure protein-protein interactions respectively.

Reagents

Human, purified, platelet derived β-thromboglobulin (comprising a group of proteins derived from the cleavage of platelet basic protein, i.e. CTAPIII, beta- thromboglobulin and NAP-2 and the main isoform of which is CTAPIII - preparation is ~95%-100% CTAPIII (Hoogewerf, A.J., et al.)), platelet factor 4, Factor V, Factor Va, Factor X, Factor Xa, and Factor IXa, prothrombin (Factor II) and alpha-thrombin (Factor I la) were purchased from Haematolgic Technologies. Normal pooled plasma, Factor VIII: C-deficient plasma, Factor IX-deficient plasma, Factor V-deficient plasma, Factor X-deficient plasma, Factor Xa chromogenic substrate (CS-01 -(65)) and Factor lla/thrombin chromogenic substrate (CS-01 -(38)) were purchased from Hyphen Biomed. Calibrated automated thrombography reagents were purchased from Thrombinoscope BV.

Mass Spectrometric Analysis

Human purified β-thromboglobulin was trypsin digested prior to analysis by mass spectrometry. The protein was incubated in 7M urea, 50mM ammonium bicarbonate, and 10mM DTT at 60°C for 1 hour, lodoacetamide (55mM, final concentration) was added and the sample was incubated for 1 hour at room temperature in the dark. The protein was diluted with 50mM ammonium bicarbonate to bring the urea concentration to 1 M. Trypsin (1 μg) was added per 20-50 μg of protein and incubated at 37°C overnight with mixing. The protein was then incubated at -20°C for 1 hour to inactivate trypsin, followed by lyophilisation by speed vacuuming at 60°C for 1 hour. The pellet was then resuspended in 20 μΙ of 98% water, 2% acetontitrile, and 0.1 % formic acid and desalted using a ZipTip μΰ18. The protein was run on a Thermo Scientific Q Exactive mass spectrometer connected to a Dionex Ultimate 3000 (RSLCnano) chromatography system. Tryptic peptides were resuspended in 0.1 % formic acid. Each sample was loaded onto a fused silica emitter (75 μιη ID, pulled using a laser puller (Sutter Instruments P2000)), packed with UChrom C18 (1.8 μιη) reverse phase media (nanoLCMS Solutions LCC) and was separated by an increasing acetonitrile gradient over 60 minutes at a flow rate of 250 nL/min. The mass spectrometer was operated in positive ion mode with a capillary temperature of 320 °C, and with a potential of 2300V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution (70,000) MS scan (300-1600 m/z) was performed using the Q Exactive to select the 12 most intense ions prior to MS/MS analysis using HCD. The raw data was de novo sequenced and searched against the Human subset of the Uniprot reviewed database using the search engine PEAKS Studio 7 (Bioinformatics Solutions), for peptides cleaved with trypsin. Each peptide used for protein identification met specific Peaks parameters, i.e. only peptide scores that corresponded to a false discovery rate (FDR) of <1 % were accepted from the Peaks PTM database search. Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT)

PT and APTT were measured using the fully automated ACL TOP coagulometer (Beckman Coulter) using PT-fibrinogen HS plus reagent and HemosIL APTT lyophilized silica reagent, respectively (Instrumentation Laboratory). Calibrated Automated Thrombography

Thrombin generation in platelet-poor plasma was assessed using a Fluoroskan Ascent plate reader (Thermo Lab System) in combination with Thrombinoscope software (Thrombinoscope BV). Briefly, 80μΙ of plasma was incubated with 20μΙ of MP reagent containing 4μΜ phospholipids (60% phosphatidylcholine, 20% phosphatidylserine, and 20% phosphatidylethanolamine) or platelet-poor plasma reagent containing 1 pM Tissue Factor and 4uM phospholipids. Thrombin generation was initiated by automatic dispensation of fluorogenic thrombin substrate (Z-Gly-Gly-Arg-AMC.HCI) and 100 mM CaCI2 into each well (final concentrations, Z-Gly-Gly-Arg-AMC.HCI, 0.42 mM and CaCI2, 16.67 mM). Thrombin generation was determined using a thrombin calibration standard. Measurements were taken at 20-second intervals for 60 minutes, or until thrombin generation was complete. The lag time to start of thrombin generation, peak amount of thrombin generated (nM of lla, time to peak thrombin (tpeak), and area under the thrombin generation curve, represented by endogenous thrombin potential (ETP), were measured.

Chromogenic clotting factor activation assays

The activation of Factor X and Factor II in a purified protein system was measured using Factor Xa and Factor lla specific chromogenic substrates, respectively. For Factor X activation, Factor Xa (100nM), Factor X (2μΜ), β- thromboglobulin (200Mg/ml), Factor X plus β-thromboglobulin (2μΜ + 200μg/ml), or Factor X plus Factor IXa (2μΜ + 200μg/ml) were incubated for 1 hour with shaking (450rpm on an orbital shaker) in a 40μΙ volume of a phosphate buffered saline solution supplemented with 3mM CaCI2. Following this, 200μΙ of Factor Xa chromogenic substrate (CS-01 -(65), 1 mg/ml) was added and the absorbance at 405nm was read after 20 minutes. For Factor II activation, Factor I la (100nM), Factor II (2μΜ), β-thromboglobulin (200Mg/ml), or Factor II plus β- thromboglobulin (2μΜ + 200μg/ml) were incubated for 1 hour with shaking (450rpm on an orbital shaker) in a 40μΙ volume of phosphate buffered saline supplemented with 3mM CaCI2. Following this, 200μΙ of Factor Xa chromogenic substrate (CS-01 -(38), 1 mg/ml) was added and the absorbance at 405nm was read after 20 minutes.

Surface Plasmon Resonance

A BIAcore X system (BIAcore, Uppsala, Sweden) was used to evaluate β- thromboglobulin binding to immobilised Factor X. Factor X was immobilised (25 μg/mL in 10 mM NaOAc, pH 4.5) onto the surface of a CM5 sensor chip at a flow rate of 10 μΙ/minute for 10 minutes. The surface was capped with 1 M ethanolamine HCI (pH 8.5) and washed with 10 mM NaOH (flow rate 10 μυι-ninute) to remove unbound or extraneous material. An uncoated flow cell was used to detect nonspecific binding, β-thromboglobulin (0-20μg/ml in hydroxyethyl piperazine ethanesulfonic acid buffered saline (HBS-EP) with 0.005% (v/v) surfactant P20, pH 7.4) and Factor IXa (0-4 g/ml in HBS-EP with 0.05% (v/v) Tween-20, pH 7.4) was passed over the Factor X surface at a flow rate of 30 μυι-ninute. Association and dissociation phases were monitored for 3 and 10 minutes, respectively. Sensograms for each β-thromboglobulin and FIXa concentration were fitted to a 1 :1 interaction model using Biacore 3000 dedicated software. The surface was regenerated using 5 mM NaOH for β-thromboglobulin and 20 mM NaOH for Factor IXa

Statistics

Data are expressed as the mean plus standard error of the mean and representative of at least 3 independent experiments. Data was analysed using GraphPad Prism software (Horsham, Pa, USA). Paired t tests or repeated measures ANOVA followed by Tukey's multiple comparison post test were used to analyse data sets. Results

Mass spectrometry confirmed the absence of TF, FVIIa, FVIIIa, FIXa, FVa, and FXa in the 3TG preparation. In normal pooled plasma, 3TG dose-dependently increased the rate and extent of TF stimulated thrombin generation. In the absence of 3TG, the lagtime was 9±1 min, which was shortened in a dose dependent manner upon incubation with 3TG (10C^g/ml; 5±1 min, p<0.05). Incubation with 3TG (5C^g/ml) also shortened the APTT (35±1 to 25±1 sees, p<0.05) and reduced the lagtime to thrombin generation (26±5 to 7±1 min, p<0.05) and increased the peak thrombin generation (60±30 to 97±23nM, p<0.05) in the absence of an exogenous TF stimulus. Immunodepleted plasmas and inhibitory antibodies were used to determine the underlying mechanism of action. In FVIII deficient plasma, thrombin generation was not observed in the absence of an exogenous TF stimulus. However, upon incubation with 3TG (5C^g/ml), thrombin generation was observed and peak thrombin generation increased from 1 ±1 to 75±12nM lla (p<0.05). 3TG also shortened the APTT in FVIII-deficient plasma from 131 ±8 to 103±14 sees (5(^g/ml, p<0.05). This procoagulant effect of 3TG was not inhibited by TF or FVIIa inhibitory antibodies, suggesting that the effect was independent of the intrinsic tenase complex, TF or FVIIa. Interestingly, homologous PF4 did not induce thrombin generation in FVIII- deficient plasma (peak thrombin generation was 75±12nM v 0±0nM lla upon incubation with 50μg/ml 3TG and PF4 respectively). The procoagulant effect of 3TG persisted when thrombin generation was independent of FV activation (supplementation of FV deficient plasma with FVa). In contrast, the effect was not observed when thrombin generation was independent of FX activation (supplementation of FX deficient plasma with FXa). Collectively, these data suggest that 3TG may modulate FX activation. To investigate this hypothesis, chromogenic FX activation was measured. Cleavage of a FXa specific chromogenic substrate was observed upon incubation of 3TG with FX, suggesting a direct effect of 3TG upon FX activation. No measurable chromogenic substrate cleavage was observed upon incubation with 3TG or FX alone. In contrast, 3TG did not induce prothrombin activation, measured using a thrombin-specific chromogenic substrate. Using SPR, 3TG was found to bind directly to immobilised FX (KD 1.36±0.7x10 ^"7 M). The kinetics of the interaction between 3TG and FX was lower than that between FIXa and FX (KD 4.25±1.90

10 ^"9 ).

Conclusion

In conclusion, 3TG has been identified as a novel platelet-derived activator of coagulation FX. Moreover, 3TG is capable of inducing thrombin generation in immunodepleted FVIII and FIX deficient plasma, a finding that is of translational relevance to patients with Haemophilia A or B.

Various modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art without departing from the scope of the invention as defined in the claims.